Methods of blocking the interaction between stromal cells and hemopoietic cells with anti-VCAM-1 antibodies

ABSTRACT

A therapeutic method of modulating the immune response, by administering to a patient an amount of IL-4 effective to promote peripheral blood lymphocyte adhesion to microvascular endothelial cells in lymphoid organs. The IL-4 is preferably coadministered with IL-1β. 
     An improved method of screening a cell line for the production of a binding partner that binds with a cell adhesion molecule, by contacting the binding partner with IL4-activated and nonactivated microvascular endothelial cells, and selecting binding partners that bind to the IL4-activated microvascular endothelial cells but not to the nonactivated microvascular endothelial cells. The selected binding partners may thereafter be tested for the ability to block lymphocyte binding to cytokine-activated endothelial cells. The binding partners are preferably also characterized by binding to human VCAM-1 and to IL4- or TNFα-activated bone marrow stromal cells. A representative embodiment is mAb 6G10 produced by hybridoma ATTC No. HB10519. 
     A therapeutic method of modulating the immune response in a patient, by administering to the patient an agent that specifically binds to IL4-activated microvascular endothelial cells, in an amount effective to impede transmigration of lymphocytes that specifically bind to VCAM-1 from blood across postcapillary venules.

This is a Rule 62 File Wrapper Continuation of U.S. application Ser. No.08/051,455, filed Apr. 21, 1993, now abandoned, which in turn is a Rule60 Divisional of U.S. patent application Ser. No. 07/156,008, filed Aug.2, 1990, and issued as U.S. Pat. No. 5,206,345 on Apr. 21, 1993.

This invention was made with government support under Public HealthService grants CA40272, P30 CA15704, and RR00166. The government hascertain rights in this invention.

TECHNICAL FIELD

This invention relates to genetic engineering involving recombinant DNAtechnology, and particularly to therapeutic methods and reagents formodulating the immune response including treating inflammation in apatient.

BACKGROUND OF THE INVENTION

The following abbreviations are used in this disclosure: CAM, celladhesion molecules; EBM, endothelial basal medium; EC, endothelialcells; EDTA, ethylene-diaminetetraacetic acid; EGF, epidermal growthfactor; ELAM-1, endothelial leukocyte adhesion molecule-1; FBS, fetalbovine serum; HEV, high endothelial venules; HUVEC, human umbilical veinendothelial cells; ICAM-1, intracellular adhesion molecule-1; IL-1,interleukin-1; IL-1β, interleukin-1-beta; IL-4, interleukin-4; INF-γ,interferon-gamma; LDL, low density lipoprotein; LFA-1, lymphocytefunction-associated antigen-1; LTBMC, long-term bone marrow culturesystem; mAb, monoclonal antibody; MLN, mesenteric lymph node; PBL,peripheral blood lymphocytes; PBS, phosphate-buffered saline; PMN,polymorphonuclear leukocyte; SEM, standard deviation; TNF-α, tumornecrosis factor-alpha; VCAM-1, vascular cell adhesion molecule-1; VLA-4,leukocyte integrin VLA-4; WM, Waymouth medium. Throughout thespecification, the notation “(#)” is used to refer to the documentslisted in the appended Citations section.

Migration of lymphocytes from the bloodstream into surrounding tissuesis a dynamic, multistep process initiated by attachment to the luminalsurface of endothelial cells (EC) lining the postcapillary venules.Certain components of the microvasculature, notably the morphologicallydistinct high endothelial venules (HEV) found in lymphoid organs such aslymph nodes. Peyer's patches, and tonsils, continuously supportlymphocyte binding and transmigration. Some adhesive interactionsattendant with movement into these sites are, at least operationally,organ-specific (1-6). Others are mediated by cell adhesion molecules(CAM) that have more general tissue distributions, for example,ICAM-1/LFA-1 interactions (7-9). During both acute and chronicinflammation, microvascular endothelial cells at other sites can beinduced to support traffic of various leukocyte subtypes (5, 10).Accumulation of lymphocytes in chronic inflammations, e.g., arthriticsynovia, is usually accompanied by conversion of the local postcapillaryvenules to a cuboidal morphology and expression of new adhesivestructures (3, 5). It has been suggested that lymphocyte adhesion toendothelial cells in chronic inflammatory lesions also incorporates anelement of organ- or site-specificity (11). The complete identity andbalance of inductive factors in the local microenvironment thatcontribute to the endothelial-cell “traffic” phenotype, and particularlyits organ-specific character at some sites, have yet to be defined inmolecular terms. Other factors are likely to be important, e.g.,endothelial cell contact with the underlying extracellular matrix (12);but, clearly, release of proinflammatory cytokines in the local milieucontributes markedly to the upregulation of cell adhesion molecules onendothelial cells (13, 14).

For example, interleukin-1 (IL-1), TNF-α, and IFN-γ have all been shownto increase adhesiveness of cultured endothelial cells for granulocytesand lymphocytes (15-23). In some cases these effects are paralleled byenhanced leukocyte migration to sites of cytokine injection in vivo (24,25). Recently, much progress has been made in identification of specificcell adhesion molecules induced on endothelial cells by proinflammatorycytokines. IL-1, for example, induces endothelial leukocyte adhesionmolecule-1 (ELAM-1), a member of the LEC-CAM (Lectin, EGF,Complement-Cellular Adhesive Molecule) family (19, 26), which isselectively adhesive for polymorphonuclear leukocytes and weaklyadhesive for monocytes. Similarly, cytokine induction of intercellularadhesion molecule-1 (ICAM-1), a ligand for the leukointegrin LFA-1(lymphocyte function-associated antigen-1), has been reported onendothelial cells (27). Recently, vascular cell adhesion molecule-1(VCAM-1) was identified as a TNF- and IL1-inducible ligand forVLA4-mediated attachment of lymphocyte adhesion to human umbilical veinendothelial cells (HUVEC) (28-30). Although not directly linkedfunctionally to lymphocyte transmigration, other cell surface markersassociated with traffic endothelium in vivo have been shown to beinduced by IFN-γ (15). Additional adhesive ligands of more limitedtissue distribution, termed vascular addressins, MECA-79 and MECA-367,have been identified in lymph nodes and mucosal lymphoid tissues,respectively (31, 32). Whether these ligands can be induced in vitro byspecific cytokines is not known at this point, but studies of transgenicmice suggest that IFN-γ may contribute to their expression in vivo (33).

The capacity of cytokines to enhance lymphocyte adhesion tomicrovascular-derived endothelial cells has been analyzed in rodents andsheep (15, 24, 25). In humans, wherein most of the molecular definitionof EC-CAM exists, cytokine induction has been studied almost exclusivelyusing umbilical vein as the endothelial cell source (16-19). As pointedout recently by Issekutz (24), certain disparities exist between resultsobtained in these systems.

Because of this and since our preliminary results indicated thatlarge-vessel- and microvascular-derived endothelial cells might differin cytokine responses via-á-vis adhesive events, we endeavored to testhow immunologically active cytokines affected lymphocyte adhesion toprimate (macaque) microvascular endothelial cells. Further, since therehave been suggestions of cytokine dependence for the traffic endothelialcell phenotype not only at sites of inflammation, but also for highendothelium in lymph nodes (34), mesenteric lymph nodes were used as onesource of microvascular endothelial cells.

SUMMARY OF THE INVENTION

Our results indicate that IL1β- and IFNγ-induced microvascularendothelial cells behave similarly to HUVEC. In contrast, IL-4 on itsown, and in combination with IL-1β, was a potent effector of lymphocyteadhesion to microvascular endothelial cells, but had only minimaleffects on umbilical-vein-derived endothelial cells. A significantportion of the increased adhesion was due to upregulated expression ofVCAM-1 or a serologically related molecule.

The invention accordingly provides, in one aspect, a therapeutic methodof modulating the immune response in a patient, by administering to thepatient an amount of IL-4 effective to promote peripheral bloodlymphocyte adhesion to microvascular endothelial cells in lymphoidorgans and thereby modulate the patient's immune response. The IL-4 ispreferably coadministered with IL-1β to the patient. In a representativeembodiment, IL-4 is administered to a patient needing treatment forinflammation, to promote transmigration of lymphocytes from blood acrosspostcapillary venules at sites of inflammation in the patient.

Another aspect of the invention is the provision of an improved methodof screening a cell line for the production of a binding partner thatbinds with a cell adhesion molecule. The method includes the steps ofcontacting the binding partner with cells bearing the cell adhesionmolecule and detecting any binding reaction between the binding partnerand the cells. The improvement involves contacting the binding partnerwith IL4-activated and nonactivated microvascular endothelial cells, andselecting cell lines that produce binding partners that bind to theIL4-activated microvascular endothelial cells but not to thenonactivated microvascular endothelial cells. As an additional screeningstep, the binding partners of the selected cell lines may thereafter betested for the ability to block lymphocyte binding to cytokine-activatedendothelial cells. In this manner, the invention provides immunologicaland peptide binding partners that specifically bind to IL4-activated butnot nonactivated microvascular endothelial cells. The binding partnersare preferably also characterized by the ability to block lymphocytebinding to cytokine-activated endothelial cells, and most preferably bybinding to human VCAM-1 and to IL4- or TNFα-activated bone marrowstromal cells. A representative embodiment of this most preferredbinding partner is mAb 6G10 produced by hybridoma ATTC No. HB 10519.

In a related aspect, the invention provides a therapeutic method ofmodulating the immune response in a patient, by administering to thepatient an agent that specifically binds to IL4-activated microvascularendothelial cells, in an amount effective to impede transmigration ofcells, such as lymphocytes or tumor cells, that specifically bind toVCAM-1 from blood across postcapillary venules into extracellular fluidin the patient.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A and 1B shows photomicrographs of representative microvascularendothelial cells cultured in serum-containing (FIG. 1A) and serum-freemedia (FIG. 1B), as described in Example 1;

FIG. 2 shows uptake of a representative endothelial-cell marker bycultured microvascular and other endothelial cells, as described inExample 1;

FIGS. 3A, 3B, 3C and 3D shows photomicrographs of lymphocyte adhesion tocultured microvascular endothelial cells following cytokine activation,as described in Example 2;

FIGS. 4A, 4B and 4C shows representative time courses ofcytokine-induced lymphocyte adhesion to cultured microvascularendothelial cells, as described in Example 2;

FIG. 5 shows stability of IL4/IL-induced lymphocyte adhesion toendothelial cells, as described in Example 3;

FIG. 6 shows lymphocyte adhesion to IL4/IL1-activated endothelial cellsis divalent cation dependent, as described in Example 4;

FIGS. 7A, 7B, 7C and 7D shows labeling of cultured microvascularendothelial cells with mAb 6G10, as described in Example 4;

FIG. 8 shows monoclonal antibody 6G10 blocks the lymphocyte adhesion toIL4/IL1-induced macaque microvascular endothelium, as described inExample 4;

FIGS. 9A, 9B, 9C and 9D shows labeling of CHO transfected cells withmAb's 6G10 and 4B9, as described in Example 4;

FIG. 10 shows that mAb 6G10 recognizes an approximately 110 kD moleculeon cytokine-activated endothelial cells, as described in Example 4;

FIGS. 11A and 11B shows IL4/TNFα-enhancement of mAb 6G10-recognizedantigen expression on bone marrow stromal cells, or described in Example5; and,

FIG. 12 shows that the VLA-4 receptor for VCAM-1 is expressed at highlevels on bone marrow cells bearing the CD34 antigen, as described inExample 5.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

Adhesion of lymphocytes to endothelial cells (EC) is the requisite firstelement in the multistep process of transmigration from blood across thepostcapillary venules. Selective expression of cell adhesion molecules(CAM) by microvascular endothelial cells in lymphoid organs (e.g., lymphnodes) and during tissue inflammation modulates this traffic in asite-directed manner. CAM synthesis by endothelial cells is regulated inturn by cytokines released in the local microenvironment. Studies donelargely with human umbilical vein endothelial cells (HUVEC) haveimplicated IL-1, IFN-γ, and TNF-α as cytokines that promote leukocyteadhesion to endothelial cells. In the work reported here, the responsesof cultured microvascular endothelial cells derived from macaque lymphnodes to IL-1β, IL-2, and IL-4 were examined. Increases in lymphocyteadhesion following preculture of microvascular endothelial cells inIL-1β or IFN-γ were typically two-to fourfold above controls andcomparable to those reported for HUVEC. IL-2 had no effect. The moststriking finding followed stimulation with IL-4. While only marginaleffects on large vessel cultured endothelial cells were seen, thiscytokine markedly enhanced adhesion to microvascular endothelial cells.IL-4 induced adhesion was observed as early as 4 hours after induction,plateaued by 24 hours, was stable through 72 hours of culture, butdecayed to basal levels within 72 hours after removal of IL-4 from thecultures. IL-1β, but not IL-2 or IFN-γ, synergistically enhanced theaction of IL-4 on cultured microvascular endothelial cells to promotelymphocyte binding. Adhesion triggered in this manner required de novoprotein synthesis. However, the avidity of IL4-activated microvascularendothelial cells for lymphocytes, and analyses of kinetics, cation andtemperature dependence, and/or lack of blockade with mAb's to ELAM-1,ICAM-1, and MECA-79 indicated that these CAM were not central to thephenomenon. To aid identification of the relevant CAM, mAb's specific toIL4-induced microvascular endothelial cells were produced. One of these,6G10, blocked up to 90% of lymphocyte adhesion to IL4-inducedmicrovascular endothelial cells and reacted specifically with CHO cellstransfected with human VCAM-1, an endothelial ligand of the β1 integrin,VLA-4. Our results indicate that IL-4 may have potent effects onlymphocyte recirculation in vivo and that endothelial cell subtypes mayregulate VCAM-1 differentially in response to specific cytokines.

Numerous studies suggest that recruitment of leukocytes to sites of bothacute and chronic inflammation is triggered by increased expression andfunction of cell adhesion molecules (CAM) on endothelium as well as onblood leukocytes (3, 10, 14). That elevated CAM function is accomplishedin part by release of proinflammatory cytokines such as IL-1, IFN-γ, andTNF-α is also well documented in the recent literature (11, 13, 23, 28).Other elements of the complex process of vessel transmigration, lateralmovement on the endothelial surface, and diapedesis, for example, areundoubtably facilitated or inhibited by other cytokines, e.g., IL-8(51). Moreover, some endothelial CAM are more adhesive in vitro forcertain leukocyte subpopulations than others (17, 19, 52). Also,kinetics of individual CAM expression may differ, and their induction byindividual cytokines may be selective. Therefore, local secretion ofthese soluble molecules could provide a means whereby the influx oflymphoid, monocytic, and granulocytic cells could be regulated with adegree of independence.

The studies reported here were undertaken because of dissimilaritiesbetween some of the in vitro data on leukocyte adhesion tocytokine-stimulated endothelium and in vivo findings on recruitment tosites of cytokine injection. For example, while IFN-γ markedlystimulates lymphocyte migration to skin after local injection (23, 53,54), significant but less striking effects on lymphocyte adhesion tocultured human endothelium were reported (20, 27). Reciprocally, IL-1,which stimulates adhesion well in vitro, did not enhance recruitment toskin at injection sites in rats (23, 54). In principle, these disparateobservations could be explained by species-specific differences sincemost in vitro studies have used human umbilical vein endothelial cellsas the cellular substrate for adhesion, while in vivo experiments havetypically utilized rodent or ovine models (24, 25). Alternatively,distinctions between microvascular and large vessel derived endothelialcells could be responsible. For example, Issekutz (24) and Hughes et al.(55) did find that IFN-γ treatment of rat microvascular endothelialcells significantly increased the binding of lymphocytes. An additionalexplanation for the disparity between in vivo and in vitro datasuggested by the work of Oppenheimer-Marks and Ziff (56) is that IFN-γpromotes both adhesion and subsequent migration across endothelium,while IL-1 may only stimulate the initial lymphocyte binding. To helpresolve some of these issues, we developed a system to examine adhesionof lymphocytes to cultured lymph node microvascular endothelium.Relatively pure cultures of endothelial cells were propagated frommacaque lymph nodes as disclosed below. These cultured cells had thecharacteristic dome-shaped, cobblestone morphology of culturedendothelial cells and expressed markers characteristic of endothelium,such as uptake of acetylated-LDL. Our results are summarized in Table 1.

TABLE 1 Effect of cytokines on cultured microvascular endothelium.Medium Cytokine(s) none IL-1β IL-2 IFN-γ Complete EBM none 44.5 ± 12.0 188 ± 21.9 46.0 ± 6.9   65.5 ± 14.8  (2% FBS) IL-4 200 ± 37.2 615 ± 74.4230 ± 57.5 251 ± 22.0 Complete CS-1.55 none 250 ± 53.7 656 ± 67.7 291 ±57.8 452 ± 36.9 (serum-free) IL-4 684 ± 48.4 1226 ± 99.3  633 ± 88.2 856 ± 107.2 Endothelial cells were treated with IL-1β (1 ng/ml), IL-2(100 u/ml), IL-4 (10 ng/ml), IFN-γ (100 u/ml), or their combination, orleft unstimulated for 24 hr as indicated. After removal of cytokines,PBL were added to each well, the adhesion assay was performed (constantagitation for 30 min at +4° C.) and lymphocytes bound to endothelialcells were counted. Each value represents a mean number of adherentlymphocytes per mm² ± SEM.

In general, the results for IL-1β, IFN-γ, and IL-2 were similar to thosereported previously for HUVEC. Both IL-1β and IFN-γ stimulatedlymphocyte adhesion, albeit more strongly and consistently in the caseof IL-1β, while IL-2 was without any noticeable effect. The moststriking finding was that IL-4 treatment of endothelial cells markedlyincreased their adhesiveness for lymphocytes. This was apparent as earlyas 4 hours after treatment, reached a plateau by approximately 12-24hours, and was maintained for at least 72 hours. The effect required denovo protein synthesis and continued presence of cytokines formaintenance. Interestingly, this robust effect of IL-4 was not observedon HUVEC, or on endothelial cells from macaque aorta; in these casesIL-4 had either no effect or only marginally enhanced lymphocyteadhesion (J. Harlan, J. Pober, personal communications; Masinovsky andGallatin, unpublished data). Typically, the maximal increase overbackground in lymphocyte adhesion was in the same range (approximatelytwofold) as that reported recently for IL4-treated HUVEC by Thornhill etal. (52). The mechanisms underlying this differential response of thetwo endothelial cell types is not known. However, it is probably notexplained by a relative absence of IL-4 receptors per se on HUVEC. Inmacaque, at least, IL-4 receptor expression of large vessel andmicrovascular endothelial cells is roughly equivalent (Masinovsky andBeckmann, unpublished data).

The involvement of LFA-1/ICAM-1 mediated adhesion, at least as anobligatory part of this phenomenon, could be excluded because theIL4-induced binding was: a) not sensitive to cold temperature, b)primarily calcium dependent, and c) not blocked by addition of mAb tothese CAM. Similar antibody inhibition tests excluded a necessaryinvolvement of MECA-79, CD44, and class II MHC in the process. Althoughnot directly excluded, the utilization of ELAM-1 as an adhesivecomponent seems unlikely. Kinetics of its induction versus the adhesionobserved here are different, and ELAM-1 is preferentially adhesive forneutrophils and monocytes rather than for lymphoid cells. The fact thatone mAb, 6G10, specifically reactive with IL4-induced microvascularendothelial cells, blocked lymphocyte adhesion and reacted selectivelywith human VCAM-1 transfectants strongly suggests that VCAM-1 or aserologically closely related molecule (of 100-110 kD, see below)mediates lymphocyte binding in this system. It might not be the onlyrelevant structure since other cell surface molecules were induced byIL-4 on microvascular endothelial cells (Masinovsky and Gallatin,unpublished data). However, the identification of these additionalcomponents, as cell-adhesion molecules, has not been established.

VCAM-1, also known as INCAM-110, was recently identified on TNF-α- andIL1-induced HUVEC (28, 29). Molecular analyses reveal that it is amember of the immunoglobulin supergene family and has as one ligand theβ1-integrin, VLA-4 (29, 30). Although its precise physiologic roleduring inflammation has yet to be defined, recruitment of lymphocytes tochronic inflammations such as arthritic synovia is one possibility.Involvement of VCAM-1, or a related molecule, in normal traffic throughmucosal lymphoid tissue may also occur since the α-chain of VLA-4 hasbeen implicated in lymphocyte binding to high endothelium in Peyer'spatches (2). VCAM1-mediated adhesion may selectively recruit onlycertain lymphocyte subsets since VLA-4 expression is not uniform on allperipheral lymphocytes. In fact, direct evidence for lymphocyte subsetbiased adhesion to cytokine activated endothelium was provided recentlyby Damle and Doyle (57). The involvement of VLA-4/VCAM-1 mediatedadhesion was not tested in their study, but this would be an intriguingpossibility that could explain their data in part. In addition to itsfunction in lymphoid traffic, VCAM-1 may also be an important factor intumor metastasis (28). Unfortunately, other mAb's currently available toVCAM-1 react poorly with nonhuman primate endothelial cells (Masinovskyand Gallatin, unpublished data; M. Bevilacqua, J. Harlan, J. Pober,personal communication). Since mAb 6G10 reacted with both human andmacaque endothelial cells it should provide a useful tool to address theissues of tissue distribution, in vivo function, and the role of IL-4 inVCAM-1 induction. For example, tests to determine if systemicadministration of IL-4 results in VCAM-1 induction could provide insightinto the mechanisms underlying vascular leakage and the lymphocytopeniaobserved during clinical therapy with this cytokine.

That IL-4 may regulate lymphocyte traffic is perhaps not surprising inhindsight. Originally described as a B cell stimulant (58), IL-4 hasreceptors on a variety of cell types, including nonhemopoietic cells(41-44), and triggers many distinct responses. IL-4 promotes adhesionbetween B and T cells, in part by upregulation of class II MHC molecules(59). The IL-4 acted synergistically with IL-1β to activatemicrovascular endothelial cells is a novel finding but not withoutprecedent. IL-4 acts together with IL-2 and IL-5 to regulate secretionof different immunoglobulin isotypes (60), with GCSF to promotegranulopoeisis (46), and with IL-3 to regulate the differentiation ofmast cells (61). Similarly, synergy between IL-1 and TNF has beenreported in inducing migration of PMNs during inflammation (62).Although antagonism between IL-4 and IFN-γ has been reported during Bcell activation (63, 64), we did not observe an effect of this type oncultured endothelial cells (Table 1). The mechanism underlying synergybetween IL-1 and IL-4 in this system is at present unknown. While notformally excluded, it seems unlikely that either cytokine exerts itseffect solely through induction of the other. When tested along over awide range of concentrations, neither cytokine induced as much adhesionas when the two were applied together. Nonetheless, it will beinteresting to determine if either IL-4 or TNF-α, which are both goodinducers of VCAM-1, act directly on endothelial cells or indirectlythrough induction of a second soluble mediator.

EXAMPLE 1 Propagation of Microvascular Endothelial Cells

To initiate this work, a procedure for obtaining uniform cultures ofendothelial cells from lymph nodes was derived. Small colonies withendothelial morphology in cultures prepared from macaque mesentericlymph nodes, as outlined in the appended Materials and Methods section,were recognized 2-3 days after primary plating. Cells had a polygonalshape with few contacts between them. By the 5th to 7th day the culturesgrew to confluency, most of the growth occurring at the periphery of thecolony, at which point the first selective removal of fibroblasts andother nonendothelial cells was carried out by limited trypsinization.When combined with selective growth media, this procedure typicallyyielded cultures which were virtually free of contamination by cellshaving a fibroblastoid morphology or markers characteristic of dendriticor monocytic cells. Although endothelial cells were successfully grownin all three of the media preparations used, the media varied in theirrelative capacities to selectively propagate endothelial cellsmaintaining typical endothelial markers and cytokine responses (seebelow). For example, morphological differences were observed betweenmicrovascular endothelial cells grown in different media, withserum-containing media giving a higher frequency of cells having acobblestone morphology characteristic of endothelial cells (FIG. 1A).Reciprocally, the most rapid and selective growth of endothelial cellscompared to nonendothelial cells was obtained with the serum-freeCS-1.55 medium (FIG. B).

Referring to FIGS. 1A and 1B in more detail, the two photomicrographs,of live cultured microvascular endothelium of macaque mesenteric lymphnodes, show the dome-shaped appearance of cells grown to confluency incomplete EBM containing 2% FBS (FIG. 1A), as compared to the moreelongated cells in serum-free endothelial cell medium, complete CS-1.55(FIG. 1B). Scale bars indicate 100 μm.

Referring to FIG. 2, uptake of acetylated-LDL, a marker associated withendothelium, by endothelial cells grown in CS-1.55 was similar to thatobserved for endothelial cells from aorta. In particular, FIG. 2 is aflow cytometric analysis of acetylated-LDL uptake by cultured macaqueendothelial cells grown in CS-1.55 from: aorta (heavy continuous line);microvascular endothelial cells of mesenteric lymph nodes (MLN) (dashedline); MLN microvascular endothelial cells activated with IL-4 (10ng/ml) and IL-1β (1 ng/ml) for 24 hrs (dotted line); andnegative-control cultured human foreskin fibroblasts (HFF) (thincontinuous line). Cells were exposed to acetylated-LDL (10 μg/ml) for 6hr, then dislodged with trypsin-EDTA, and analyzed on a Coulter EPICS720-2 flow cytometer; see Materials and Methods.

Endothelial cells grown in serum-free medium were passaged successfullymore than six times without any apparent morphological changes. Incontrast, usually after the third passage, microvascular endothelialcells grown in the presence of serum assumed a more flattened shape,frequency losing contacts with the substrate. Accordingly, forserum-containing cultures, only cells from the first two passages wereused for adhesion assays.

EXAMPLE 2 Lymphocyte Adhesion to Cultured Cytokine-Induced MicrovascularEndothelial Cells

The capacities of IL-1β, IL-2, IL-4, and IFN-γ to induce endothelium tobe more adhesive for lymphocytes was examined as follows. Briefly,cultures of microvascular endothelial cells from macaque mesentericlymph nodes were cultured in the presence of these cytokines for varyinglengths of time, washed, and assayed for lymphocyte adhesion asdescribed below (see Materials and Methods). Monocytes were removed fromthe lymphocyte suspension prior to assay. The results are shown in Table1 and FIGS. 3A, 3B, 3C, 3D, 4A, 4B and 4C.

FIGS. 3A, 3B, 3C and 3D shows photomicrographs of PBL adhesion tocultured endothelial cells from mesenteric lymph nodes of Macacanemestrina. Endothelial cells were grown to confluency and activated bycytokines for 24 hr. Following the adhesion assay, cells were fixed with1% glutaraldehyde in PBS. The FIGS. 3A, 3B, 3C and 3D, respectivelyindicate endothelial cell activation with: (a) IL-1β (1 ng/ml); (b) IL-4(10 ng/ml); (c) IL-1β (1 ng/ml) and IL-4 (10 ng/ml); and (d) control, nointerleukins added. Arrows indicate adherent lymphocytes; arrowheads,endothelial cells; scale bar, 100 μm.

FIGS. 4A, 4B and 4C plots adhesion of PBL to cytokine-activated culturedmicrovascular endothelial cells at different cytokine concentrations andincubation times (4 hours, 24 hours and 72 hours, respectively). Beforeeach adhesion assay, endothelial cells were treated with cytokines(solid circles, IL-4; open circles, IL-1β; triangles, IL-4 in thepresence of 1 ng/ml of IL-1β) for the time indicated, which incubationwas followed by the adhesion assay. Lymphocyte binding to theseendothelial cells was determined by visual count of attachedlymphocytes. Each point represents the mean number of attached PBL±SEM.

As anticipated from previous reports (16, 18, 20) after 24 hrs ofinduction, both IL-1β and IFN-γ treatments yielded similar increases inthe number of adherent lymphocytes. Stimulation indices of 2 to 4compared to controls were typical for cytokine concentrations in therange of 1 pg/ml to 10 ng/ml (FIGS. 3A, 3B, 3C and 3D and Table 1). IL-2had no effect on lymphocyte binding at any concentration tested (Table1, and data not shown).

In contrast to the rather minimal effects observed with the othercytokines, IL-4 treatment consistently yielded the most dramaticincrease in lymphocyte adhesion (FIGS. 3A, 3B, 3C, 3D, 4A, 4B and 4C andTable 1). The level of PBL adhesion to IL4-induced endothelial cellsexceeded that of the control (no cytokines) by up to 45-fold (FIG. 4B).The optimum concentration of IL-4 that promoted abundant adhesion oflymphocytes to endothelial cells was in the range of 100 pg/ml to 10ng/ml; this optimum was common for all time periods (4, 24, and 72 hrs)of endothelial cell exposure to this cytokine (FIGS. 3A, 3B, 3C, 3D, 4A,4B, and 4C). At these concentrations, the absolute numbers oflymphocytes bound to IL4-exposed endothelial cells were consistently inthe range of 200-1300 lymphocytes/mm², while on the control endothelialcells only 5-40 lymphocytes/mm² were observed. The simulation index(which ranged from 2 to 45) varied among different animals used as asource for microvascular endothelium, and was lower for endothelialcells propagated in serum-free medium, due essentially to the higherbackground adhesion on endothelial cells cultured in this manner (Table1).

IL-4 promoted adhesion by acting on microvascular endothelial cellsrather than on lymphocytes, since addition of an anti-IL-4 antibody tothe cultures during the assay, or addition of IL-4 to lymphocytesuspensions for 30 min prior to assay, did not affect the lymphocytebinding to cultured endothelial cells. IL4-induced adhesion was detectedas early as 4 hr after cytokine treatment, plateaued by 12-24 hr, andremained in evidence through at least 72 hr in the continued presence ofIL-4 (FIGS. 4A, 4B and 4C). The effect of IL-4 was relatively specificto cultured microvascular endothelial cells, since addition of thiscytokine to other adherent cells, such as fibroblasts, which havefunctional receptors for IL-4 (41-44), did not result in increasedlymphocyte adhesion in our system (data not shown). At the highesttested concentrations of IL-4 (50-100 ng/ml) and of IL-1β (5-10 ng/ml),cultured microvascular endothelial cells underwent morphologicalchanges, converting from a cobblestone phenotype to a more flattenedappearance with fewer cell-cell contacts and a detachment of cells fromthe plates. These changes were apparent after 24 hrs of cytokineexposure and resulted in decreased lymphocyte adhesion per unit area ofthe culture well.

EXAMPLE 3 Synergism Between IL-4 and IL-1 in Promoting LymphocyteAdhesion to Cultured Microvascular Endothelial Cells

Because IFN-γ, IL-1β, and IL-4 all enhanced adhesion to microvascularendothelial cells, and since these cytokines had been reported tosynergize with other interleukins in promoting various hemopoeiticactivities (45, 46), we tested their effect on microvascular endothelialcells in combination. After 24 hours of costimulation the effects ofIL-4 and IFN-γ were at best additive with no significant synergy inevidence (Table 1). Similarly, additive but not synergistic activity wasobserved when IL-1β and IFN-γ were applied in conjunction (data notshown). In contrast, the amount of lymphocyte adhesion observed whenboth IL-4 and IL-1β were added to the microvascular endothelial cellcultures was markedly increased over that expected from purely additiveeffects (FIGS. 3A, 3B, 3C, 3D, 4A, 4B and 4C, Table 1). When the amountof IL-1β was held constant (0.1 or 1 ng/ml) and varying amounts of IL-4were added, synergistic activation of cultured endothelial cells wasobserved over a wide range of cytokine concentrations (FIGS. 4A, 4B and4C). In the presence of as little as 1 pg/ml of IL-4, addition of IL-1βincreased lymphocyte adhesion 2.5 to 7 times compared to that detectedwith IL-4 alone (FIGS. 4A, 4B and 4C). For lower concentrations of IL-1β(0.1 ng/ml), synergistic lymphocyte adhesion was approximately half ofthat observed at 1 ng/ml. Addition of IL-1β did not significantly alterthe kinetics of the response to IL-4, nor did it change the optimumconcentrations of IL-4 that promoted adhesion (FIGS. 4A, 4B and 4C). Thesuper-additive effect of IL-1β and IL-4 was manifest only under certainculture conditions. For endothelial cells grown in serum-free medium,CS-1.55, which typically had background adhesion 5-10 fold higher thanthat observed in other media, the effects of these two cytokines wereonly additive (Table 1).

Referring to FIG. 5 in more detail, after 24 hr incubation with IL-4 (1ng/ml) and IL-1β (1 ng/ml), the cytokines were removed. The adhesionassay was conducted after additional incubation without any lymphokines.Each point (open circles) represents the mean adherence±SEM of eightreadings. Control endothelial cells (solid circle) received no cytokineadded.

The combinatorial effect of IL-1β and IL-4 on lymphocyte binding tomicrovascular endothelial cells required de novo protein synthesis. Ifafter 2 hr of cytokine activation with IL-1β (1 ng/ml) and IL-4 (10ng/ml), the metabolic inhibitor, emetine, was added, adhesion observed 2hr later was inhibited to 10% of that seen without addition of emetine.In similar experiments, if emetine was added after 70 hr of endothelialcell activation, adhesion measured at 72 hr was unaffected compared tocontrols. Once induced, the adhesive function of endothelial cells wasrelatively stable over time. Maintenance of the relevant cell adhesionmolecules in a functional form did depend, however, on continuedpresence of the cytokines. When endothelial cells were incubated withIL-1β and IL-4 for 24 hr, and then the interleukins were removed, thecapacity to support lymphocyte adhesion was almost unchanged during thefirst 8 hr, decayed by 50% at 24 hr, and returned to essentiallybackground levels by 72 hr (FIG. 5).

EXAMPLE 4 Identification of One CAM Involved in IL-4 Induced LymphocyteAdhesion to Microvascular Endothelial Cells

A priori, IL-4's effects in this system could be manifest throughinduction of known adhesion molecules, or might involve expression ofnovel CAMs. To examine these possibilities, the following approacheswere taken. First, the temperature dependence and cation requirements oflymphocyte binding were determined. Temperature dependence was minor,with robust adhesion observed at both 4° and 37° C. (FIGS. 3A, 3B, 3C,3D, 4A, 4B and 4C, Table 1, and data not shown). Also, lymphocytebinding was divalent cation dependent, with a primary requirement forcalcium rather than magnesium ions (FIG. 6). Both of these facts tend toexclude LFA-1 (CD11/CD18/CAM-1 mediated interactions since these aresensitive to low temperature and utilized magnesium as the preferredcation (47).

Referring to FIG. 6 in detail, endothelial cells were activated withIL-1β (1 ng/ml) and IL-4 (10 ng/ml) for 24 hr. Subsequently, endothelialcells were fixed in 1% paraformaldehyde in PBS, washed, and the adhesionassay was conducted in PBS-based medium containing 1% BSA and 1% glucoseto which Ca⁺⁺ or Mg⁺⁺ were added as shown. Each bar represents the meannumber of adherent lymphocytes±SEM of four readings. Closed bars:IL1/IL4-activated endothelial cells; open bars: control, no cytokineadded.

Secondly, the sensitivity of IL-4 induced adhesion to blockade by mAbknown to interfere with cell-cell interactions mediated by the CAMs,CD44, MECA-79, and by interactions between LFA-1 and ICAM-1 was testedby pretreating the relevant cell (i.e., lymphocyte or endothelial cell)with each mAb prior to assay (see Materials and Methods). Although allthese reagents reacted well with macaque endothelial cells and/orlymphocytes in culture or in tissue sections, none of the interferedsignificantly with lymphocyte binding under these conditions. Sinceupregulation of class II MHC molecules on endothelium had been reportedafter induction with IFN-γ (50-52), an anti-class II MHC mAb, Hb10a, wasalso tested and likewise did not effect lymphocyte attachment (data notshown). A similar assessment could not be made for ELAM-1 since theavailable antibodies against this molecule did not react well withmacaque endothelial cells. Nonetheless, other differences between ELAM-1mediated adhesion and that observed following IL-4 stimulation wereinconsistent with involvement of this particular CAM in a primary role(see above). In parallel experiments conducted with two differentantibodies (4B9 and E1/6) against human VCAM-1 (aka INCAM-110 (28-30)),only weak reactivity with cytokine-induced microvascular endothelialcells was detected by immunofluorescent confocal microscopy (data notshown). However, in one experiment out of four conducted, an inhibition(40%) of lymphocyte binding to IL4/IL4-induced microvascular endothelialcells was detected. Although inconclusive, this result suggested thatVCAM-1 might contribute to the process of attachment, especially sincemAb's 4B9 and E1/6 were relatively species specific. These mAb's reactedpoorly in immunocytochemical tests of macaque tissues.

To resolve the issue, mAb's specific to IL-4 induced macaque endothelialcells were prepared (see Materials and Methods). Briefly, hybridomasderived from fusions using spleen cells from mice immunized withcytokine-stimulated endothelial cells were first screened by ELISA forselective reactivity with IL4/IL1-activated endothelium. Positive cloneswere tested secondarily for their ability to block lymphocyte binding toendothelial cells under various conditions of cytokine treatment. OnemAb elicited in this manner, 6G10, detected on cultured endothelialcells a cell-surface antigen whose expression paralleled the amount oflymphocyte adhesion induced by each cytokine (i.e.,IL1β+IL-4>IL-4>IL-1β, FIGS. 7A, 7B, 7C and 7D). More importantly, 6G10blocked up to 80% of lymphocyte adhesion occurring after endothelialcell treatment with IL-1β/IL-4 (FIG. 8). To clarify the identity of thestructure recognized by mAb 6G10, this reagent was tested by indirectimmunofluorescence for reactivity with CHO cells transfected with cDNAsencoding human ELAM-1, ICAM-1, VCAM-1, or CD4 as a control. We foundthat mAb 6G10, similar to mAb 4B9, bound specifically with transfectantsexpressing the product of the VCAM-1 gene (FIGS. 9A, 9B, 9C and 9D). Noreactivity was observed on the other transfectants. By extension then,lymphocyte binding to IL-4 stimulated endothelial cells in this systemmost probably utilizes the macaque homologue of human VCAM-1 or aserologically related molecule as a major adhesive component.

Referring to FIGS. 7A, 7B, 7C and 7D in detail, endothelial cells wereactivated for 24 hr with: (FIG. 7A) control, no cytokine added; (FIG.7B), IL-1β (1 ng/ml); (FIG 7C) C, IL-4 (10 ng/ml); and (FIG. 7D), IL-4(10 ng/ml) and IL-1β (1 ng/ml). After stimulation, unfixed cells wereexposed to mAb 6G10 for 2 hr at room temperature, which was followed byexposure to biotinylated secondary antibody, avidin-FITC labeling, andanalysis on Bio-Rad laser scanning confocal microscope. Note an increaseof immunofluorescence with IL-4 and IL-1β+IL-4 activation of endothelialcells. Scale bar is 50 μm.

Referring to FIG. 8, endothelial cells were activated with IL-4 (10ng/ml) alone or in a combination with IL-1β (1 ng/ml) for 24 hr. Afterremoval of cytokines, cells were exposed to either mAb 4B9 or 6G10 for30 min, and the adhesion assay was conducted. Each bar represents meannumber of adherent lymphocytes±SEM of four readings.

FIGS. 9A, 9B, 9C and 9D shows immunofluorescence of: (FIG. 9A) humanVCAM-1 transfectant labeled with 6G10; (FIG. 9B) same as in (FIG. 9A)but labeled with 4B9; FIG. 9C, human ICAM-1 transfectant labeled with6G10; and (FIG. 9D) human ELAM-1 transfectant labeled with 6G10. Scalebar is 100 μm.

FIG. 10 shows radioimmunoprecipitation of cell surface molecules ofmicrovascular EC with mAb 6G10. EC were grown to confluency in completeEBM and were either activated with IL-4 (10 ng/ml), TNF-60 (10 ng/ml),or IL-4 (10 ng/ml) and TNF-α (10 ng/ml), or served as a controlreceiving no cytokines. EC were labeled with ¹²⁵I, lysed,immunoprecipitated with mAB 6G10, and electrophoresed on a 10% SDS gelunder reducing conditions. Note distinct band at 110 kD at lanes 2-4,which is absent in the control lane.

EXAMPLE 5 IL-4 and TNF Induce mAb 6G10-Recognized Expression on BoneMarrow Stromal Cells

Thirteen years ago, Dr. Michael Dexter and his colleagues (65)established the methodology for maintaining the survival and developmentof primitive bone marrow stem cells over long periods of time in vitro.This so-called long-term bone marrow culture (LTBMC) system, whileinitially optimized for the growth of murine cells, has subsequentlybeen modified to support the growth of human bone marrow (66). Theessential feature of both systems is the development of an adherentlayer of mesenchymal cells derived from the stromal cell population ofthe bone marrow. The inductive influences provided by the stromalelements of these cultures are essential for the growth and self-renewaland the differentiation of stem cells in these cultures to morespecialized progeny (e.g., myeloid progenitors) in a manner whichreflects the in vivo situation. Both cytokines released by the stromalcells and adhesive interactions between stromal cells and hemopoieticprecursors are important in this process (67, 68). At this point, manyof the specific adhesive mechanisms utilized in this in vitro system andits in vivo counterpart are ill-defined.

We have discovered that the antigen recognized by mAb 6G10 is expressedon human bone marrow stromal cells in vitro especially after inductionwith IL-4 and/or TNF. FIGS. 11A and 11B shows human bone marrow stromalcells grown in long-term marrow culture according to established methodsfor 2 weeks. In this representative experiment, the cultures weretreated for 25 hr with recombinant human TNF-α and IL-4 (10 ng/ml) priorto immunolabeling with mAb 6G10 (20 μg/ml) FIG. 11A or isotype-matchedcontrol antibody FIG. 11B and goat anti-mouse IgG-FITC (SouthernBiotechnology Assoc.). Immunofluorescence images were recorded using ascanning laser confocal microscope. The IL4/TNFα-enhancement of6G10-recognized antigen expression on the stromal cells is evident inFIG. 11A. This novel finding would not have been predicted a priori fromavailable information about the tissue distribution of VCAM-1.Interestingly, mAb 4B9 (from John Harlan), which also recognizes VCAM-1on human endothelium, did not bind significantly to human bone marrowstroma cultured in this manner. Therefore, the antigenic epitoperecognized by mAb 6G10 may be unique compared to that bound by mAb 4B9.Cell-surface molecules immunoprecipitated with mAb 6G10 fromTNFα/IL4-activated cultured human bone marrow stroma differed from thatobserved on activated omentum EC. The 6G10-recognized molecules werelarger in size—one of the species having a molecular weight of 115-130kD, while the other was larger than 200 kD—as compared to the 100 kD oftraditional VCAM-1.

Further, we have discovered that a major receptor for VCAM-1, VLA-4(also known as integrin alpha4/beta1 (69)), is expressed at high levelson bone marrow cells bearing the CD34 antigen. FIG. 12 shows confirmingFACS plots of human CD34⁺ bone marrow cells stained with negativecontrol mAb (thin line), and anti-VLA-4 mAb 163H (thick line). Thisfinding of coexpression is significant because CD34 expressiondistinguishes a subset of bone marrow cells (1-4%) which are enriched inprimitive stem cells and progenitors (70). Therefore, we infer thatadhesive interactions within the bone marrow between hemopoietic stemcells and/or progenitor cells and stromal elements may be mediated bythe binding of VLA-4 and the antigen recognized by 6G10. It has beendemonstrated in a large animal system (canine) and in man thatantibodies directed against two known adhesion molecules, CD44 andLFA-1, respectively (71, 72, 73), administered before or during bonemarrow transplantation facilitate the transplantation process in caseswhere the grafted marrow and recipient are not perfect genetic matches.

It follows that mAb 6G10 and its antigen-binding derivatives, as well asother hybridoma-generated or recombinantly engineered binding partnershaving antigen-binding specificities like mAb 6G10, VCAM-1 and itsderivatives, particularly those recognized by mAb 6G10, and VLA-4 andits derivatives are useful either in vitro or in vivo to modify ineither a positive or negative fashion the growth or differentiation ofbone marrow stem cells or bone marrow stroma. For example, the disclosedcytokines and reagents are useful to facilitate the success of bonemarrow transplantation, either by enhancing the growth of the graft(e.g., by IL-4 or TNF-α administration), or by preventing graft versushost disease (e.g., by pretreatment with soluble VCAM-1 receptorrecognized by mAb 6G10, or antibodies directed to the VLA-4 receptorrecognized by the antigen recognized by mAb 6G10).

The disclosed reagents can also be used to immunoselect (e.g., by FACScell sorting, magnetic bead selection, or negatively by complementlysis) primitive hemopoietic stem cells, progenitor cells, or bonemarrow stromal elements.

The above-stated applications are particularly useful in cases where thedisclosed cytokines are administered either in vivo or in vitro topromote bone marrow transplantation as a curative regimen for neoplasticdisease or anemia.

The disclosed reagents are readily conjugated to radionuclides or otherpharmaceutical moieties to provide targeting devices to achieve highspecific localization of the radionuclides or pharmaceutical agents tothe bone marrow, for purposes of radioimaging or therapy of neoplasticdisease, anemia, or benign hyperplasias of hemopoietic origin.

Since IL-4 and TNF-α induce 6G10 antigen expression on bone marrowstroma, the coordinated use of either cytokine in vivo with injection ofthe aforementioned antibody or receptor conjugates can achieve enhancedspecificity of antibody or receptor conjugate localization to the bonemarrow.

MATERIALS AND METHODS Endothelial Cell Cultures

Mesenteric lymph nodes of 0.5- to 7-year-old Macaca nemestrina wereobtained through the Tissue Distribution Program of the Regional PrimateResearch Center at the University of Washington, Seattle, Wash. Thelymph nodes were transported in an ice-cold washing solution composed ofRPMI-1640 medium buffered with 25 mM HEPES, pH 7.0, and containingtylosin and gentamycin (Sigma). For separation of small blood vesselsand endothelial cells, a modification of the method described byWilliams (35) was used. Briefly, lymph nodes were cut into pieces andtransferred to a digestive solution containing 0.5 mg/ml collagenaseType IV (Sigma), 0.5 mg/ml dispase (Boehringer-Mannheim), and 0.05 mg/mlDNAse (Boehringer-Mannheim) in complete Waymouth medium (WM), pH 7.2,containing 10% heat inactivated fetal bovine serum (FBS, HyClone) andpenicillin/streptomycin (GIBCO). The tissue was incubated at 37° C. andagitated by vortexing every 15-20 minutes. Collections of suspended,single cells and small cell aggregates were performed every 0.5-1 hr.Small clumps of cells, fragments of capillaries and larger vessels wereseparated from the majority of lymphocytes and individual stromal cellsby brief centrifugation at 200xG. Pellets were washed in the washingsolution and resuspended in either complete WM, or in completeEndothelial Basal Medium (EBM, Clonetics) with 2% FBS, or in serum-freeendothelial cell medium CS-1.55 (Cell Systems) with endothelial cellgrowth supplement (ECGS, 10 μg/ml, Cell Systems) and heparin (50 μg/ml,Cell Systems). Cells were cultured at 370° C. with 5% CO₂ in 25 cm²flasks (Falcon) coated previously with 1% gelatin (Sigma) in phosphatebuffered saline (PBS), pH 7.0. Approximately 18 hr later, unattachedcells were aspirated with fresh medium added. Growing fibroblasts wereremoved either mechanically with a scraper, or by a 90 sec digestionwith 1x trypsin/EDTA (Gibco) on day 5-10. This procedure was repeatedevery 3-7 days. When endothelial cells grew to confluence, cells weredislodged by a treatment with 1× trypsin/EDTA and passaged at a 1:3ratio into new flasks. Only cultures in which at least 95% of cells hadmorphology and markers characteristic of endothelial cells (e.g., uptakeof acetylated low density lipoprotein, lack of cytokeratin expression)were used for subsequent experiments. Under these conditions, cellshaving morphological and immunohistological markers characteristic ofmonocytic or dendritic cells were rarely seen in the cultures.

For adhesion assays, cells from the first two passages were plated at5×10³/well on gelatin-coated 24-well plates (Costar) or uncoated 24-wellPrimaria™ plates (Falcon) and incubated until they reached confluency,usually for one week. After removing spent medium, 1 ml/well of freshmedium alone or containing a cytokine, either human recombinant IL-1β,IL-2, IL-4 (Immunex), or IFN-γ (Alpha Therapeutic), or a combination ofthese was added, and cells were grown for an additional 4-72 hrs. Othersources of human recombinant IL-4 and IL-1β include Research &Diagnostic Systems, Minneapolis, Minn. In experiments to test dependencyon protein synthesis, emetine (Sigma) was added at a final concentrationof 50 μM prior to the adhesion assay.

Low Density Lipoprotein (LDL) Uptake

Cells were incubated with acetylated low density lipoprotein labeledwith dioctadecyltetramethylindocarbocyanine perchlorate (DiI-Ac-LDL,Biomedical Technologies) at 10 μg/ml for 4 hrs at 37° C. Following 2washes with PBS, cells were dislodged with trypsin-EDTA, fixed with2paraformaldehyde in PBS, and analyzed using 555 nm excitation on aCoulter EPICS 750-2 flow cytometer. A total of 2×10⁴ events werecollected as list mode files and reprocessed using Reproman™ software(TrueFACS) using forward-angle light scatter to exclude dead cells.Endothelial cells from macaque aorta grown in complete CS-1.55, andhuman foreskin fibroblasts (a gift from T. Brown, FHCRC), served aspositive and negative controls, respectively.

Adhesion Assays

Peripheral blood lymphocytes (PBL) of Macaca nemestrina were separatedfrom other blood elements by centrifugation on a discontinuous Ficollgradient as described previously (39). To remove macrophages and otherhighly adherent leukocytes, cells were incubated in complete WM at 3×10⁶cell/ml on regular tissue culture Petri dishes (Falcon) for 1-2 hrs at37° C.

For adhesion assays, endothelial cells on 24-well plates were washedtwice with ice-cold adhesion buffer (HEPES buffered RPMI-1640, pH 6.8,with 1% FBS and 1% glucose); 0.4 ml of adhesion buffer was aliquoted perwell; and plates were placed on a gyratory shaker (New Brunswick) at 50rpm at 4° C. Lymphocytes were collected from Petri dishes, counted,resuspended in ice-cold adhesion buffer at 2.5×10⁶ cell/ml, and added toendothelial cell monolayers (0.2 ml/well). After 30 min, cells werefixed by gentle addition of 2% glutaraldehyde in PBS to each well. Aftertwo washes with PBS, lymphocyte adhesion was quantified by lightmicroscopy according to the following procedure. For enumeration, eachwell was divided into four equivalent sectors. Within each sector thenumber of lymphocytes adhering to endothelial cells was determined for amicroscopic field corresponding to 0.24 mm² of the well. Results areexpressed as a mean±standard deviation. In experiments to test divalentcation dependence, endothelial cells were paraformaldehyde-fixed priorto assay, washed in PBS; and the assays were carried out in PBS with 1%bovine serum albumin, 1% glucose, with and without the addition of Ca⁺⁺(1 mM) or Mg⁺⁺ (1 mM).

Production of mAb Against Cytokine-Induced Endothelial Cells

For immunizations of BALB/C mice (Jackson Laboratories), culturedmicrovascular endothelial cells were activated with IL-4 (10 ng/ml) andIL-1β (1 ng/ml) for 24 hrs, washed with PBS, and dislodged with ascraper. After centrifugation at 200×G, the pellet was resuspended inPBS (0.5 ml/mouse) containing adjuvant peptide, muramyl dipeptide (50μg/mouse, Sigma), and cells (1-5×10⁶/mouse) were injected into micesubcutaneously (neck region) and intraperitoneally. Four subsequentboosts were conducted over a period of 8 months. Four days after thelast boost, the spleen was removed, and lymphocytes were fused with NS-1myeloma cells using polyethylene glycol (MW-1500, Aldrich) as describedby Kennett (40). Selection of hybridomas was accomplished withaminopterin (Sigma). ELISA screening of supernatants was conducted onIL4/IL1-activated versus nonactivated microvascular endothelial cells,after which positive clones were tested in the adhesion assay for theirability to block lymphocyte binding to cytokine-activated endothelialcells. Selected hybridomas were subcloned at least three times. For theproduction of pure monoclonal antibodies (mAb's), hybridomas were grownin serum-free medium (Nutridoma-NS, Boehringer-Mannheim) andimmunoglobulin was precipitated with ammonium sulfate.

The hybridoma that produces mAb 6G10 was deposited on Aug. 2, 1990,under accession No. HB 10519 at the American Type Culture Collection(ATCC), 12301 Parklawn Drive, Rockville, Md., U.S.A.

Monoclonal and Polyclonal Antibodies

For blocking lymphocyte adhesion to endothelial cells, all mAb's wereadded to either endothelial cells or lymphocytes at a finalconcentration of 10-50 μg/ml or 50% supernatant in the adhesive bufferand incubated for 30 minutes at 37° C. After 2 washes, cells were usedin the adhesion assay as described above. For immunofluorescence tests,endothelial cells or CHO cells transfected with cDNAs encoding humanVCAM-1, ICAM-1, ELAM-1, or CD4 (gifts of R. Lobb and J. Harlan) werepropagated on tissue culture 8-chamber slides (VWR). Live, or ice-coldacetone or paraformaldehyde-fixed (2% in PBS) cells were preblocked with3% goat serum in PBS, and incubated with mAb or polyclonal antibodiesfor 2 hr at room temperature. After washes, cells were incubated withFITC-labeled goat anti-mouse Ig antibodies for 30 min at roomtemperature, washed and mounted for analysis by immunofluorescentmicroscopy using a Bio-Rad laser scanning confocal microscope and imageanalysis software. The following antibodies were used: anti-ICAM (RR1/1,a gift from R. Rothlein), anti-LFA-1 (60.3, a gift from P. Beatty),anti-CD44 (Hutch-1), anti-lymph node addressin (MECA-79, a gift of P.Streeter and E. Butcher), anti-class II MHC (HB10a, a gift of E. Clark),anti-factor VIII (Calbiochem), anti-IL-4 (Immunex), and anti-humanVCAM-1 (4B9, a gift of J. Harlan; and E1/6, a gift of M. Bevilaqua). Incontrast to cultured endothelial cells from macaque aorta, microvascularendothelial cells showed no detectable labeling by polyclonal antibodiesto factor VIII, which was in complete agreement with reports of negativelabeling with factor-VIII antibodies of cultured microvascularendothelial cells from rat peripheral nodes (36-38).

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While the present invention has been described in conjunction with apreferred embodiment, one of ordinary skill in the art after reading theforegoing specification will be able to effect various changes,substitutions of equivalents, and alterations to the subject matter setforth herein. It is therefore intended that the protection granted byLetters Patent hereon be limited only by the definitions contained inthe appended claims and equivalents thereof.

1. A method of blocking interaction between a bone marrow stromal cellexpressing VCAM-1 and a cell expressing VLA-4, which comprisesadministering an antibody to VCAM-1 in an amount effective to decreaseVCAM-1 mediated adhesion between the bone marrow stromal cell and thecell expressing VLA-4.
 2. The method of claim 1 wherein the antibody toVCAM-1 is selected from the group consisting of monoclonal antibodiesand antigen-binding fragments of said monoclonal antibodies thatspecifically bind to an epitope recognized by 6G10 monoclonal antibodyproduced by hybridoma ATTC No. HB
 10519. 3. The method of claim 1wherein the cell expressing VLA-4 is a hemopoietic cell expressing CD34antigen.
 4. The method of claim 1 wherein the cell expressing VLA-4 is ahemopoietic stem cell or a hemopoietic progenitor cell.